The long-term objective of this proposal is to illustrate the mechanisms and functions of tumor suppressor gene p33ING1 in regulation of cellular senescence and organismal aging. This work will be a collaboration with the laboratories of Ruvkun, Avruch and Alexander-Bridges. The work from Ruvkun's lab showed that the longevity of C. elegans is at least partly regulated by a pathway involving daf-2, an insulin receptor-like gene, and age-1, a homologue of the mammalian phosphatidylinositol-3-OH kinase (PI3 kinase) catalytic subunit. We hypothesize that the aging of mammalian animals is regulated by a similar pathway involving homologues the genes in the C. elegans age-1 pathway. In a screen designed to identify phosphatidylinositol (3,4,5)- triphosphate (PtdIns(3,4,5)P3 binding proteins, we found that PtdIns(3,4,5)P3 can bind specifically to p33ING1, a mammalian tumor suppressor gene product. p33ING1 has been shown to interact physically with p53 in mediating cellular growth control and senescence. We propose that regulation of p33ING1 by PI3 kinase plays an important role in regulating cell proliferation and senescence. p33ING1 may be a critical missing link between the growth factor signal transduction pathways mediated through PI3 kinase and cell proliferation and senescence regulated by tumor suppressor genes. Specific Aim 1 is to determine the functional interaction between PI3 kinase and p33ING1 in mediating cellular growth control and senescence. Our hypothesis is that the activation of Pi3 kinase suppresses p33ING1 activity and its ability to induce cellular senescence and apoptosis. Specific Aim 2 is to characterize the p53/p33ING1 complex and the role of PtdIns(3,4,5)P3 binding in complex formation. We would like to determine the proteins associated with p53/p33ING1 complex and the role of PI3 kinase in the interaction. Specific Aim 3 is to determine the downstream events regulated by p33ING1. We would like to determine whether p33ING1 acts upstream or in a parallel pathway of the mammalian homologues of DAF-16. Specific Aims 4 is to determine the in vivo functions of p33ING1 by generation and characterization p33ING1 mutant mice generated by gene-targeting. We would like to generate mutant mice expressing a null allele or a constitutively active mutant allele of p33ING1. These works will illustrate the role of p33ING1 in mediating cellular senescence in culture and organismal aging in vivo.